TW201522368A - Pro-drug antibodies against tissue factor pathway inhibitor - Google Patents

Abstract

This disclosure provides pro-drug antibodies which specifically bind to tissue factor pathway inhibitor (TFPI) only after exposure to proteases from the coagulation cascade. The pro-drug antibodies described are useful for treating bleeding disorders such as hemophilia. When used in such treatments, these pro-drug antibodies show increased half-life relative to other anti-TFPI antibodies, while mitigating the potential for side effects such as thrombosis.

Description

Anti-tissue factor pathway inhibitor prodrug antibody

According to 37 C.F.R.1.821(c), this paper submits a sequence listing created on March 15, 2013 in the form of an ASCII text file named "BAYRP0004USP1_ST25.txt" and has a size of ~71 kilobits. The contents of the aforementioned archives are incorporated by reference in their entirety.

The technical field is related to the treatment of hemophilia and other coagulopathy.

Blood coagulation is a process in which blood forms a stable blood clot and stops bleeding. This process involves some zymogens circulating in the blood as well as procofactors (or "coagulation factors"). These zymogens and cofactors interact through several pathways through which they are sequentially or simultaneously converted into an activated form. Finally, this process causes thrombin to be activated by thrombin by activation factor X (FXa) in the presence of factor Va, ionic calcium and platelets. Activated thrombin in turn causes platelet aggregation and converts fibrinogen to fibrin, while fibrin is cross-linked by activated factor XIII (FXIIIa) to form a blood clot.

The process of activating Factor X can occur through two different pathways: a contact activation pathway (previously known as an intrinsic pathway) and a tissue factor pathway (previously known as an extrinsic pathway). The coagulation cascade was previously thought to consist of two equally important pathways linked to a common pathway. It is now known that the primary pathway for the initiation of blood coagulation is the tissue factor pathway. Factor X can be activated by tissue factor (TF) in combination with activating factor VII (FVIIa). The complex of factor VIIa with its major cofactor (TF) is an effective initiator for the coagulation cascade.

The tissue factor pathway of coagulation is negatively regulated by tissue factor pathway inhibitors ("TFPI"). TFPI is a natural FXa-dependent feedback inhibitor of the FVIIa/TF complex. It is a member of the multivalent Kunitz-type serine protease inhibitor. Physiologically, TFPI binds to activated Factor X (FXa) to form a heterodimeric complex, which in turn interacts with the FVIIa/TF complex to inhibit its activity, thus shutting down the tissue factor pathway of coagulation. In principle, blocking TFPI activity restores FXa and FVIIa/TF activity, thereby prolonging the duration of action of the tissue factor pathway and amplifying the production of FXa, which is a common defect in hemophilia A and hemophilia B.

Of course, some preliminary experimental evidence indicates that blocking TFPI activity by antibodies against TFPI can result in prolonged prolonged clotting time or shortened bleeding time. For example, Nordfang et al. demonstrated that prolonged prothrombin time of hemophilic plasma was routinely normalized after plasma treatment with antibodies against TFPI (Thromb. Haemost., 1991, 66(4): 464-467). Similarly, Erhardtsen et al. demonstrated that bleeding time in a rabbit model of hemophilia A is significantly shortened by anti-TFPI antibodies (Blood Coagulation and Fibrinolysis, 1995, 6: 388-394). These studies suggest that inhibition of TFPI by anti-TFPI antibodies may be useful for the treatment of hemophilia A or B. Only a few anti-TFPI antibodies were used in these studies.

Monoclonal antibodies against recombinant human TFPI (rhTFPI) can be prepared and identified using fusion tumor technology. See Yang et al. , Chin . Med. J., 1998, 111(8): 718-721. The effect of individual antibodies on dilution of prothrombin time (PT) and activated partial thromboplastin time (APTT) was tested. Experiments have shown that anti-TFPI monoclonal antibodies shorten the digested prothrombin clotting time of Factor IX deficient plasma. It suggests that the tissue factor pathway plays an important role not only in physiological coagulation but also in hemorrhagic hemophilia (Yang et al. , Hunan Yi Ke Da Xue Xue Bao, 1997, 22(4): 297-300). .

U.S. Patent No. 7,015,194 to Kjalke et al. discloses a composition comprising FVIIa and a TFPI inhibitor comprising a plurality of strains or monoclonal antibodies or fragments thereof for use in treating or preventing a bleeding event or coagulation therapy. It is also disclosed that the use of the composition in normal mammalian plasma reduces clotting time. It is further suggested that Factor VIII or a variant thereof can be incorporated into the disclosed compositions of FVIIa and TFPI inhibitors. Groups that do not suggest FVIII or Factor IX and TFPI monoclonal antibodies Hehe. In addition to the treatment of hemophilia, it also suggests that TFPI inhibitors (including multi-drug antibodies or monoclonal antibodies) can be used to treat cancer (see U.S. Patent No. 5,902,582 to Hung).

Therefore, there is a need for an improved antibody specific for TFPI for the treatment of hematological diseases and cancer.

Thus, according to the present invention, there is provided an antibody comprising (a) a first variable domain comprising a first light chain and a first heavy chain variable region, the first variable domain being immunologically bound to tissue factor pathway inhibition a reagent (TFPI); (b) masking domains attached to the amine end of the first light chain and/or the first heavy chain variable region; and (c) a protease cleavable linker inserted into the first light Between the strand and/or the first heavy chain variable region and the masking domain. The protease cleavable domain can be a thrombin, plasmin, Factor Vila or Factor Xa cleavage site. The masking domain can comprise a second variable domain comprising a second light chain and a second heavy chain variable region. The antibody may be IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE antibodies. The antibody can be a human or humanized antibody, and/or a single chain antibody. The antibody may be bivalent and comprise two masking domains, one linked to the amine terminus of each first light chain variable region, or bivalent and comprising two masking domains, one linked to each first heavy chain variable region Amino terminus, or bivalent and comprising four masking domains, one linked to the respective first light chain variable region and the amine terminal of each first heavy chain variable region, for example, wherein the two masking domains are second One light chain variable region and the two mask domains are a second heavy chain variable region, wherein the second light chain and heavy chain variable regions form a second variable domain. The second variable domain can bind to tissue factor (TF), red blood cells, or albumin. The masking domain can be an albumin binding protein. This antibody binds to the Kunitz domain 2 of the human tissue factor pathway inhibitor.

Also provided is a performance vector comprising a coding region as described above and which is under the control of a promoter, and a cell comprising the expression vector. Also provided is a pharmaceutical formulation comprising an antibody as described above formulated with a pharmaceutically acceptable buffer, carrier or diluent.

In another embodiment, a method of treating a blood coagulation disorder in an individual comprising administering to the individual an amount of an antibody sufficient to promote coagulation in the individual, the antibody comprising (a) a first variable domain, including the first a light chain and a first heavy chain variable region, the first variable domain being immunologically Binding to a tissue factor pathway inhibitor (TFPI); (b) a masking domain linked to the amine-terminal end of the first light chain and/or the first heavy chain variable region; and (c) a protease cleavable linker, inserted A light chain and/or a first heavy chain variable region and a masking domain. The protease cleavable domain can be a thrombin, plasmin, Factor Vila or Factor Xa cleavage site. The masking domain can comprise a second variable domain comprising a second light chain and a second heavy chain variable region. The antibody may be IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE antibodies. The antibody can be a human or humanized antibody, and/or a single chain antibody. The antibody may be bivalent and comprise two masking domains, one linked to the amine terminus of each first light chain variable region, or bivalent and comprising two masking domains, one linked to each first heavy chain variable region Amino terminus, or bivalent and comprising four masking domains, one linked to the respective first light chain variable region and the amine terminal of each first heavy chain variable region, for example, wherein the two masking domains are second One light chain variable region and the two mask domains are a second heavy chain variable region, wherein the second light chain and heavy chain variable regions form a second variable domain. The second variable domain can bind to tissue factor (TF), red blood cells, or albumin. The masking domain can be an albumin binding protein. The individual can be a human or a non-human mammal. The individual may have trauma, hemophilia (eg, type A or type B) or cancer. The antibody can be administered systemically or locally or regionally to the site of bleeding. The antibody can be administered subcutaneously, intravenously or intraarterially. This antibody binds to the Kunitz domain 2 of the human tissue factor pathway inhibitor.

It is contemplated that any of the methods or compositions described herein can be practiced using any of the other methods or compositions described herein.

When the word "one or one" is used in the scope of the patent application and/or the description, it may mean "one", but it is also associated with "one or more", "at least one" and "one or one" The meaning of the above is the same.

It is contemplated that any of the specific examples discussed in this specification can be practiced using any of the methods or compositions of the invention, and vice versa. Additionally, the compositions and kits of the present invention can be used to achieve the methods of the present invention.

Throughout this application, the term "about" is used to indicate that the value includes the inherent variability of the error of the device or method used to determine the value, or the variability present in the subject.

The following drawings form a part of this specification and are included to further illustrate particular aspects of the invention. The invention may be more readily understood by reference to one or more of the drawings.

Figure 1. A specific example of an anti-TFPI prodrug antibody and an illustration of how it functions in vivo.

Figure 2. Illustrative illustration of an effective masking strategy for anti-TFPI prodrug antibodies.

Figure 3A-B. Vector map of TF-bound prodrugs in which the anti-TFPI and the variable regions of TF are linked back and forth. (Fig. 3A) Vector map of the HC1-pTTF5 gA200 anti-TFPI prodrug antibody fragment. (Fig. 3B) Vector map of LC1-pTTF 641 anti-TFPI prodrug antibody fragment.

4A-C. Vector map of a TF-binding prodrug to RBC-bound prodrug, wherein the anti-TF or RBC scFV is linked to the amine terminus of the anti-TFPI antibody heavy chain. (FIG. 4A) Vector map of pQM1-3E10sc-gA200HC anti-TFPI prodrug antibody fragment. (Fig. 4B) Vector map of pQM1-T119sc-gA200HC anti-TFPI prodrug antibody fragment. (Fig. 4C) Vector map of pQM1/gA200LC anti-TFPI prodrug antibody fragment.

Figure 5A-B. Vector map of albumin-bound prodrugs. (FIG. 5A) Vector map of pQM1-56E4-gA200H anti-TFPI prodrug antibody fragment. (FIG. 5B) Vector map of pQM1-56E4-gA200L anti-TFPI prodrug antibody fragment.

Figure 6. 3E10-scFv-gA200 and Ter119-scFv-gA200 anti-TFPI IgG were stained with Coomassie and did not use dithiothreitol (DTT) under non-reducing conditions and SDS-PAGE using DTT under reducing conditions.

Figure 7. TFPI binding ELISA map to determine the binding affinity of 56E4-gA200 relative to native gA200.

Figure 8. Diagram of binding of Ter119sc-gA200 to RBC as a function of antibody concentration.

9. FIG prodrugs different anti-TFPI antibodies and unmodified anti-TFPI antibodies in humans gA200 respect TFPI serum albumin (HSA) or presence BIACORE ^TM measurements of the percentage of the binding of FIG absence.

Figure 10A-G. (Figure 10A) A plot of peak thrombin as a function of antibody concentration against anti-TFPI prodrug antibody 56E4-gA200, and anti-TFPI antibody gA200. (FIG. 10B) A graph of thrombin generation as a function of different concentrations of anti-TFPI prodrug antibody 56E4-hA200, and anti-TFPI antibody gA200, showing thrombin concentrations generated at various antibody concentrations. (Fig. 10C) The prodrug TPP-2654 was activated by thrombin and FXa activation as compared to the thrombin generation profile of its parent antibody gA200. The ability of thrombin to activate TPP-2654 is assessed by the addition of exogenous thrombin followed by hirudin and subsequent activation of the added thrombin. The control includes a reaction in which a buffer is added instead of thrombin. (Fig. 10D) shows the thrombin titration concentration test required to activate the prodrug TPP-2654. The thrombin concentration tested is within the range that physiology is likely to reach. (Fig. 10E) Indirect assessment of the ability of FXa to activate the prodrug TPP-2654. FXa and thrombin levels are increased by increasing the concentration of TF used to initiate the TGA response. The relative contribution of FXa-activated prodrugs can be measured by comparing the ability of exogenous thrombin to enhance the TPP-2654 response with those achieved by FXa and thrombin activation. (Fig. 10F) shows thrombin generation of the prodrug TPP-2652, which is activated by thrombin alone. In this titration study, the prodrug TPP-2652 requires ~2.5 U/mL thrombin to convert to active TFPIAb. (Fig. 10G) The relative insensitivity of TPP-2652 to FXa can be observed in the results of the TF titration experiments. TPP-2652 showed a small increase in thrombin generation with higher TF dose rates relative to TPP-2654, which was designed to be activated by FXa and thrombin activation (Figure 10G compared to Figure 10E) ). Figure 11. A graph of the effect of different concentrations of albumin against antibodies to TFPI prodrugs and unmodified anti-TFPI antibodies.

Figure 12. Heavy and light chain sequences that can be combined to make an anti-TFPI prodrug antibody of the invention.

Figure 13. Heavy and light chain sequences of anti-TFPI prodrug antibodies of the invention.

Figure 14. Amino terminal sequence of selected heavy and light chains of an anti-TFPI prodrug antibody in accordance with the present invention.

Figure 15a. TFPI binding of albumin-binding anti-TFPI prodrugs (surface plasmon resonance-Biacore data) in the absence or presence of human or monkey albumin; Figure 15b. In the absence or presence of human/monkey albumin, Albumin binds to TFPI binding (surface plasma resonance) of anti-TFPI prodrugs after treatment with or without thrombin or FXa.

Figure 16. Mass spectrum of anti-TFPI prodrug TPP-2652 and TPP-2654 after protease cleavage.

The present disclosure describes anti-tissue factor pathway inhibitors (TFPI) for safe and long-acting antibodies for hemophilia and other therapies. Currently, anti-TFPI antibodies are in preclinical and clinical development, respectively, but the in vivo half-life of anti-TFPI antibodies is relatively shorter than the half-life of other IgG antibodies. This may be due to the removal of the target medium. In addition, anti-TFPI antibodies have also been proposed to cause side effects in patients with inflammation or treatment with FVIIa.

In order to solve these problems, the anti-TFPI prodrug antibody described in the present invention has been developed. These antibodies significantly reduce binding to TFPI before they are exposed to proteases produced from the coagulation cascade. Once coagulation begins and proteases are produced, the protease activates the anti-TFPI antibody by cleavage of the masking domain, thereby increasing its binding to TFPI. These prodrug antibodies can be used to treat bleeding disorders such as hemophilia while providing better safety and pharmacokinetic profiles than the previously described anti-TFPI antibodies.

1. Anti-TFPI prodrug antibody

The antibodies disclosed herein specifically bind to TFPI; that is, they bind to TFPI with a high affinity (e.g., at least twice as high) as their binding affinity for an incoherent antigen (e.g., BSA, casein). The term "tissue factor pathway inhibitor" or "TFPI" as used herein means any variant, isoform and species homolog of human TFPI which is naturally expressed by a cell.

In some embodiments, the prodrug antibodies of at least about 10 ⁵ M ^-1 to about 10 ¹² M ^-1 (e.g. ^{105M -1, 10 5.5 M -1,} 10 6 M -1, 10 6.5 M -1, 10 7 M ^-1 , 10 ^7.5 M ^-1 , 10 ⁸ M ^-1 , 10 ^8.5 M ^-1 , 10 ⁹ M ^-1 , 10 ^9.5 M ^-1 , 10 ¹⁰ M ^-1 , 10 ^10.5 M ^-1 , 10 ¹¹ M ^- The affinity of ¹ , 10 ^11.5 M ^-1 , 10 ¹² M ^-1 ) is bound to TFPI. Any method known in the art to analyze the antibody binds to an antigen affinity (K _d), by any method known in the art including, for example, immunoassays (such as a specific enzyme-linked immunosorbent analysis (ELISA)), bimolecular interactions Analysis (BIA) (for example, Sjolander &Urbaniczky; Anal . Chem. 63: 2338-2345, 1991; Szabo, et al. , Curr. Opin. Struct . Biol. 5: 699-705, 199, incorporated herein by reference) , and fluorescence activated cell sorting (FACS) to quantify antibodies that bind to cells expressing antigen. Now biospecific interaction analysis technique (e.g., BIACORE ^TM) BIA is a case where the marker without any interaction thereof. Optical phenomena Surface plasma resonance (SPR) changes can be used as an indicator of immediate response between biomolecules.

Anti-TFPI prodrug antibodies can use a substantially full length immunoglobulin molecule (eg, IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA), antigen binding fragments thereof (such as Fab or F(ab') ₂ ), or a construct comprising an antigen binding site (such as scFv, Fv or diabody), which is capable of specifically binding to TFPI. The term "antibody" also includes other protein scaffolds capable of directing the insertion of antibody complementarity determining regions (CDRs) into the same active binding configuration found in native antibodies such that binding to TFPI is observed in such chimeric proteins. The TFPI-binding activity of the native antibody from which the CDRs are derived.

As used herein, an "isolated antibody" is an antibody that is substantially free of other antibodies having different antigenic specificities (eg, a single antibody that binds to TFPI is substantially free of antibodies that bind to an antigen other than TFPI). However, an isolated antibody that binds to an epitope, isoform or variant of human TFPI may be cross-reactive with other related antigens, such as from other species (eg, TFPI species homologs). The isolated antibody may be substantially free of other cellular material and/or chemicals.

Specific anti-TFPI antibodies are disclosed in U.S. Patent Publication Nos. US2012/20268917, US 2012/0108796, US 2011/0229476, and International Patent Publication No. WO 2012/135671, the entire disclosure of each of each of

A. Masked Antibodies

In some embodiments, the prodrug antibodies disclosed herein are engineered to have a masking domain that reduces the ability of the antibody to bind to TFPI. These masking domains can identify elements of the coagulation cascade or other related markers. In some embodiments, the masking domain includes elements that recognize the following biomolecules, such as tissue factor (TF), red blood cells (RBC), and/or albumin. These masking domains are attached to the variable region of the antibody via a protease cleavage site as shown in Figure 1. These masking domains can be resistant Body, peptide, protein, or another scaffold. In any event, the masking domain prevents the antibody from binding to the TFPI through its variable region until the masking domain is removed.

The antibodies of the prodrugs disclosed herein are engineered to include a protease cleavage site that is recognized by one or more proteases, the cleavage of which releases the masking domain and allows the antibody to bind to the TFPI. As used herein, "protease cleavage site" means an amino acid sequence that is recognized and cleaved by a protease. In some embodiments, a protease cleavage site is positioned to mask the variable region of an anti-TFPI antibody and is shown in Figure 1. In some embodiments, the anti-TFPI prodrug antibody comprises one or more protease cleavage sites that can be cleaved by thrombin, fibrinolytic enzyme, and/or factor Xa. It is also envisioned that other protease cleavage sites for proteases that are activated or upregulated by the coagulation cascade can be used. In some embodiments, the amino acid sequence that masks the variable region of the anti-TFPI prodrug antibody comprises a polypeptide linker in addition to a protease cleavage site and/or antibody, peptide, protein, or other scaffold (as shown, For example, in Figure 1), it binds to TF, RBC, or albumin. The linker can be a single amino acid or polypeptide sequence (e.g., up to 100 amino acids). For example, the linker can be GGGGS (SEQ ID NO: 149). Other linkers that are available include those shown in SEQ ID NOs: 151-176. In other embodiments, no linker is present, and the cleavage site itself is masked by binding to TF, RBC or albumin antibodies, peptides, proteins, or another scaffold as shown in Figure 1 to bind to TFPI. Insert into the variable area.

The optimal cleavage site for at least two thrombin has been determined: (1) X ₁ -X ₂ -PRX ₃ -X ₄ (SEQ ID NO: 147), wherein X ₁ and X ₂ are hydrophobic amino acids and X Both ₃ and X ₄ are non-acidic amino acids; and (2) GRG. The thrombin is specifically cleaved after the arginine residue. The plasmin also cleaves the above two cleavage sites, but has a lower specificity than thrombin. Other thrombin cleavage sites available for use are provided as SEQ ID NOs: 1-60. Other available fibrinolytic enzyme cleavage sites are provided as SEQ ID NOs: 12, 47, 48, 53, and 61-130. In some embodiments, the cleavage site is LVPRGS (SEQ ID NO: 137).

In some embodiments, a Factor Xa cleavage site is used, such as I-(E or D)-G-R (SEQ ID NO: 148). Other Factor Xa cleavage sites available for use are provided as SEQ ID NOs: 29, 59, and 61-69.

In addition to the cleavage site, a second binding site for the protease (a so-called outer binding site) can be introduced into the anti-TFPI prodrug to create a more efficient cleavage. The outer binding site of thrombin may be derived from a natural outer binding site of a protease receptor or inhibitor such as PAR1, fibrinogen, and hirudin. The outer binding site may also be a derivative of other extracellular binding sites.

B. Antibody synthesis

Anti-TFPI prodrug antibodies can be produced synthetically or recombinantly. Some techniques are available for the production of antibodies. For example, phage-antibody techniques can be used to generate antibodies (Knappik et al., J. Mol. Biol. 296: 57-86, 2000, which is incorporated herein by reference). Another method for obtaining antibodies is to screen DNA libraries from B cells as described in WO 91/17271 and WO 92/01047, both incorporated herein by reference. Phage libraries are produced in these methods in which members display different antibodies on their outer surface. Antibodies are typically displayed as Fv or Fab fragments. Phage display antibodies are screened for affinity enrichment by binding to selected proteins. Antibodies can also be prepared using a three-source hybridoma methodology (e.g., Oestberg et al., Hybridoma 2 : 361-367, 1983; U.S. Patent No. 4,634,664; U.S. Patent No. 4,634,666, the entire disclosure of which is incorporated herein by reference.

Antibodies can also be purified from any cell that exhibits such antibodies, including any host cell that has been transfected with a display construct encoding the antibody. The host cell can be cultured under conditions such that the antibody is expressed. Purified antibodies can be isolated from other cellular components (e.g., certain proteins, carbohydrates, or lipids) that can associate with the antibody in the cells using methods well known in the art. These methods include, but are not limited to, size screening chromatography, ammonium sulfate discrimination, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. The purity of the formulation can be assessed by any method known in the art, such as SDS-polyacrylamide gel electrophoresis. Preparation of purified antibodies can comprise more than one type of antibody.

Alternatively, an anti-TFPI prodrug antibody can be chemically synthesized using its amino acid sequence (such as by direct peptide synthesis using solid phase techniques) (eg, Merrifield, J. Am . Chem . Soc . 85 : 2149-2154, 1963; Roberge et al., Science 269 : 202-204, 1995, both incorporated herein by reference. Protein synthesis can be carried out using manual techniques or by automation. Depending on the case, fragments of the antibody can be separately synthesized and chemically combined to produce a full length molecule.

In some embodiments, an anti-TFPI prodrug antibody can also be constructed in a "single-chain Fv (scFv) format" in which a protease cleavage site is inserted or surrounded by a peptide linker in a manner that masks its ability to recognize TFPI. , antibody, peptide, protein, or another scaffold. Because peptide linkers are necessary to maintain the two variable regions of the scFv together for antigen binding, cleavage of the peptide linker or flanking region allows the protease of interest to not activate or down-regulate the binding of the scFv to its antigen.

In some embodiments, an anti-TFPI prodrug antibody is constructed in an "IgG format" having two binding sites and capable of masking its ability to recognize TFPI in such a manner as to include one, two, three or four A protease cleavage site between the variable region and an antibody, peptide, protein or another scaffold. In each case, the protease cleavage site can be a linker on one or both sides. Furthermore, in each case the cleavage sites may be the same or different.

2. Polynucleotide

The invention also provides polynucleotides encoding prodrug antibodies. Such polynucleotides can be used, for example, to produce large quantities of antibodies for therapeutic use.

The cDNA molecule encoding the antibody can be produced using standard molecular biotechnology using mRNA as a template. Thereafter, cDNA molecules can be used in the art and are disclosed, for example, in Sambrook, et al. , (Molecular Cloning: A Laboratory Manual (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY; 1989) Vol. 1-3. Molecular biotechnology in the manual, incorporated herein by reference), is reproduced. Amplification techniques such as PCR can be used to obtain additional copies of the polynucleotide. Alternatively, synthetic chemistry techniques can be used to synthesize polynucleotides encoding anti-TFPI prodrug antibodies.

In order to present a polynucleotide encoding an antibody, the polynucleotide can be inserted into a expression vector containing elements necessary for transcription and translation of the inserted coding sequence. Expression vectors containing sequences encoding the antibody and appropriate transcriptional and translational control elements can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, et al. (1989) and Ausubel, et al. , (Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995), each incorporated herein by reference.

A variety of expression vector/host systems are available for inclusion and expression of the sequences encoding the antibodies. These include, but are not limited to, microorganisms, such as recombinant phage, plastid or cosmid DNA expressing vector-transformed bacteria; yeast transformed by yeast expression vectors; insect cells infected with viral expression vectors (eg, baculovirus) System; a plant cell system transformed with a viral expression vector (eg, broccoli mosaic virus, CaMV; tobacco mosaic virus, TMV); or a bacterial expression vector (eg, Ti or pBR322 plastid), or an animal cell system.

Control elements or regulatory sequences are those non-translated regions of the vector (enhancer, promoter, 5' and 3' untranslated regions) that interact with host cell proteins to perform transcription and translation. Such elements may differ in strength and specificity. Depending on the vector system and host, any number of suitable transcriptional and translational elements can be used, including constitutive and inducible promoters. For example, an inducible promoter can be used when colonizing in a bacterial system. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from plant cell genomes (eg, heat shock, RUBISCO and storage protein genes), or promoters or enhancers (eg, viral promoters or leader sequences) from plant viruses can be selected for In the carrier. In mammalian cell systems, promoters derived from mammalian genes or from mammalian viruses can be used. If it is necessary to produce a cell line comprising multiple copies of the nucleotide sequence encoding the antibody, a SV40 or EBV-based vector with appropriate selectable markers can be used.

3. Method for preparing TFPI antibody

A general article describing other available molecular biotechnologies, including the preparation of antibodies, is Berger and Kimmel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.); Sambrook, et al. , (Molecular Cloning). : A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY; 1989) Vol. 1-3); Current Protocols in Molecular Biology, (FMAusabel et al. [Eds.], Current Protocols, a Joint venture between Green Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 2000); Harlow et al., (Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988), Paul [Ed. ]); Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al. (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)), all incorporated herein by reference.

Genetic engineering has been used to generate murine, chimeric, humanized or fully human antibodies to generate therapeutic antibodies for human disease. Murine monoclonal antibodies have been shown to have therapeutic applications because of their short serum half-life, inability to cause human effector functions, and the production of human anti-mouse antibodies. Brekke and Sandlie, "Therapeutic Antibodies for Human Diseases at the Dawn of the Twenty-first Century," Nature 2, 53, 52-62 (January 2003). Chimeric antibodies have been shown to cause human anti-chimeric antibody responses. The humanized antibody further minimizes the mouse component of the antibody. However, fully human antibodies completely avoid the immunogenicity associated with murine elements. In particular, the use of antibodies with murine components or murine sources, such as the use of anti-TFPI monoclonal antibodies to treat chronic prophylactic treatment of hemophilia, has a high risk of developing an immune response to the therapy, as frequent administration is required and The treatment lasts for a long time. For example, antibody therapy for hemophilia A may require weekly dosing for a patient's lifetime. This is a constant stimulus for the immune system. Therefore, there is a need for fully human antibodies for antibody therapy for hemophilia and hereditary and acquired coagulopathy or defects.

Therapeutic antibodies have been made by the fusion tumor technique described by Koehler and Milstein in "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity," Nature 256, 495-497 (1975). Fully human antibodies can also be produced recombinantly in prokaryotes and eukaryotes. For therapeutic antibodies, it is preferred to recombinantly produce antibodies in host cells rather than fusion tumors. Recombinant manufacturing has the benefit of higher product consistency, higher production throughput, and controlled production to reduce or eliminate the presence of animal derived proteins. For these reasons, it is desirable to produce a monoclonal anti-TFPI antibody in a recombinant manner.

A monoclonal antibody can be produced by expressing in a host cell a nucleotide sequence encoding a variable region of a monoclonal antibody according to a specific example of the present invention. A nucleic acid containing a nucleotide sequence can be transfected and expressed in a host cell suitable for production with the aid of a performance vector. Therefore, it is also mentioned Provided is a method for producing a monoclonal antibody that binds to human TFPI, comprising: (1) transfecting a nucleic acid molecule encoding a monoclonal antibody of the present invention into a host cell, and (b) cultivating the host cell to be in the host cell A monoclonal antibody is expressed in the middle, and the produced monoclonal antibody is isolated and purified as appropriate (c), wherein the nucleic acid molecule comprises a nucleotide sequence encoding the monoclonal antibody of the present invention.

In one embodiment, in order to represent an antibody or antibody fragment thereof, a DNA encoding a partial or full-length light chain and heavy chain obtained by standard molecular biology techniques is inserted into an expression vector such that the genes are operably linked to transcription and translation. Control sequence. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector function as intended to regulate the transcription and translation of the antibody gene. The expression vector and the expression control sequence are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into an individual vector or, more typically, two genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation at complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if there is no restriction site). The light and heavy chain variable regions of the antibodies described herein can be used to operably link the _VH segment to the C by inserting them into an expression vector that has encoded the heavy chain constant region and the light chain of the desired isotype. _H and V _L segment within the vector segment is operatively linked to the C _L segment, either antibody to create full-length antibody genes isotype. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is ligated in-frame to the amine terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain encoding gene, the recombinant expression vector of the present invention also has regulatory sequences that control the expression of the antibody chain gene in the host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be understood by those skilled in the art that the design of the expression vector (including selection of regulatory sequences) can depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. Examples of regulatory sequences for mammalian host cell expression include directing in mammalian cells High-level protein-expressing viral elements, such as those derived from giant cell virus (CMV), simian virus 40 (SV40), adenovirus (eg, adenovirus major late promoter (AdMLP)), and polyomavirus promoters and/or Enhancer. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the beta-globin promoter can be used.

In addition to antibody chain genes and regulatory sequences, recombinant expression vectors can carry additional sequences, such as sequences that regulate replication of the vector in a host cell (eg, a source of replication) and a selectable marker gene. The selectable marker genes are useful for screening host cells into which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, all to Axel et al.). For example, a marker gene can generally be screened for conferring resistance to a drug (eg, G418, hygromycin or methotrexate) to a host cell into which the vector has been introduced. Examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells in the case of methotrexate selection/amplification) and the neo gene (for G418 screening).

For the expression of light and heavy chains, expression vectors encoding heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express an antibody of the present invention in a prokaryotic or eukaryotic host cell, it is preferred to express the antibody in eukaryotic cells and to express the antibody optimally in a mammalian host cell because of such eukaryotic cells (especially It is a mammalian cell that assembles and secretes correctly folded and immunologically active antibodies more than prokaryotic cells.

Examples of mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220 In combination with DHFR selectable markers, for example as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, HKB11 cells and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibodies are produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods such as ultrafiltration, size screening chromatography, ion exchange chromatography, and centrifugation.

4. Use partial antibody sequences to represent intact antibodies

The antibody interacts with the target antigen primarily through amino acid residues located on the six heavy and light chain CDRs. To this end, the amino acid sequences within the CDRs between individual antibodies are more diverse than sequences outside the CDRs. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express mimetic-specific natural antibodies by constructing expression vectors that are ligated into skeletal sequences of different antibodies with different properties from CDR sequences from different specific natural antibodies. Recombinant vectors (see, for example, Riechmann, L. et al. , 1998, Nature 332: 323-327; Jones, P. et al. , 1986, Nature 321:522-525; and Queen, C. et al. , 1989 , Proc. Natl. Acad. Sci. USA 86: 10029-10033). Such framework sequences can be obtained from public DNA databases, including germline antibody gene sequences. These germline sequences will differ from the mature antibody gene sequences as they will not include fully assembled variable region genes, which are formed by V(D)J junction during B cell maturation. It is not necessary to obtain the complete DNA sequence of a particular antibody to reproduce an intact recombinant antibody having binding properties similar to those of the original antibody (see WO 99/45962). Part of the heavy and light chain sequences spanning the CDR regions are generally sufficient for this purpose. Partial sequences were used to determine which germline variable and zygote segments contributed to the recombinant antibody variable gene. The germline sequence is then used to fill the missing portion of the variable region. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. For this reason, the corresponding germline leader sequence used to represent the construct must be used. For the sequence to which the deletion is to be added, the selected cDNA sequence can be combined with the synthetic oligonucleotide by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized into a set of short, overlapping oligonucleotides that are combined by PCR amplification to create a fully synthetic variable region selection strain. This process has certain advantages, such as elimination or inclusion or specific restriction sites, or specific codon optimization.

The nucleotide sequences of the heavy and light chain transcripts are used to design a set of overlapping synthetic oligonucleotides to create a synthetic V sequence that has the same amino acid coding ability as the native sequence. The synthetic heavy chain and kappa chain sequences may differ from the native sequence in three respects: the repeated nucleotide base strings are interrupted to facilitate oligonucleotide synthesis and PCR amplification; the optimal translation initiation site is based on The Kozak rule (Kozak, 1991, J. Biol. Chem. 266:19867-19870) was incorporated; and the HindIII site was engineered upstream of the translation initiation site.

The two heavy and light chain variable regions, the optimized coding, and the corresponding non-coding strand sequences are disrupted to a portion of 30-50 nucleotides at approximately the midpoint of the corresponding non-coding oligonucleotide. Thus, for each strand, the oligonucleotides can be assembled into overlapping double strands spanning 150-400 nucleotide segments. These pools are then used as templates to generate PCR amplification products of 150-400 nucleotides. Typically, a single variable region oligonucleotide set will be disrupted into two pools and separately amplified to produce two overlapping PCR products. These overlapping products are then combined by PCR amplification to form a complete variable region. It is also possible to incorporate overlapping fragments comprising heavy or light chain constant regions upon PCR amplification to generate fragments that can be readily cloned into expression vector constructs.

The reconstituted heavy and light chain variable regions are then combined with the selected promoter, translation initiation, constant region, 3' non-translated, polyadenylation, and transcription termination sequences to form an expression vector construct . The heavy and light chain expression constructs can be combined into a single vector, co-transfected, continuously transfected or separately transfected into a host cell and then fused to form a host cell that expresses both strands.

Thus, in another aspect, structural features of human anti-TFPI antibodies (e.g., TP2A8, TP2G6, TP2G7, TP4B7, etc.) are used to create a structurally similar human anti-TFPI antibody that retains the function of binding to TFPI. More specifically, one or more CDRs of a monoclonal antibody of the invention that specifically recognize the heavy and light chain regions can be recombinantly combined with known human framework regions and CDRs to create additional recombinantly engineered inventions. Human anti-TFPI antibody.

Accordingly, in another embodiment, a method for producing an anti-TFPI antibody, comprising: preparing an antibody comprising: (1) a human heavy chain framework region and a human heavy chain CDR, wherein the human heavy chain CDR3 comprises a selected from the group consisting of Amino acid sequence SEQ ID NO: 388-430 amino acid sequence, and/or (2) human light chain backbone region and human light chain CDR, wherein the light chain CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: The amino acid sequence of 259-301; wherein the antibody retains the ability to bind to TFPI. In other embodiments, the method is practiced using other CDRs of the invention.

5. Pharmaceutical composition

The anti-TFPI prodrug antibody can be provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is preferably pyrogen free. A pharmaceutical composition comprising an anti-TFPI prodrug antibody can be administered alone or in combination with at least one other agent, The agent is, for example, a stable compound which can be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose and water. A variety of aqueous carriers can be employed, such as 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter. These solutions can be sterilized by conventional sterilization techniques known (e.g., filtration). The compositions may optionally contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions such as pH adjusting agents and buffers. The concentration of the anti-TFPI prodrug antibody in the pharmaceutical composition can vary widely, ie, less than about 0.5%, usually at or at least 1% to as high as 15% or 20% by weight, and is primarily based on fluid volume, viscosity. Etc., select according to the specific investment mode selected. See, for example, U.S. Patent No. 5,851,525, incorporated herein by reference. More than one different anti-TFPI prodrug antibody can be incorporated into the pharmaceutical composition if desired.

In addition to the active ingredient, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers including excipients and auxiliaries for use in the pharmaceutical compositions to facilitate the processing of compositions into preparations. The pharmaceutical composition can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, non- Intestinal, topical, sublingual or rectal.

After the pharmaceutical compositions have been prepared, they can be placed in a suitable container and labeled for treatment of the indicated condition. Such labels will include the quantity, frequency of administration, and method. The composition may also be further encapsulated in a kit comprising one or more containers that are constrained together by a suitable packaging material, including styrofoam and a plastic blow molded container, including instructions for storage and use, as appropriate.

6. Methods for treating bleeding disorders A. Illness

Hemophilia is a hereditary condition that impairs the body's ability to stop bleeding to control blood clotting or coagulation when the blood vessels are damaged. Hemophilia A (factor VIII deficiency) is the most common form of this condition, with one in every 5,000-10,000 male infants. Hemophilia B (coagulation factor IX deficiency) is one in every 20,000-34,000 male infants.

Like most recessive X-chromosome disorders, hemophilia is more likely to occur in men than in women. This is because women have two X chromosomes, and there is only one male, so a defective gene will definitely be expressed in the male with it. Because women have two X chromosomes, hemophilia is rare, and women with two defective copies are at a low rate, so women are almost asymptomatic carriers of almost the condition. Female takers can inherit a defective gene from their mother or father, or they may be a new mutation. Although women are not unlikely to have hemophilia, this is not common: a woman with hemophilia A or B must be a hemophilia male and a female daughter with the original, and because of coagulation Factor XI deficiency, non-sex hemophilia C has an effect on both sexes, more common in the Ashkenazi Jewish (East European) offspring, but less common in other ethnic groups.

Hemophilia reduces plasma coagulation factor levels of coagulation factors required for normal coagulation processes. Therefore, temporary sputum is formed when the blood vessel is injured, but the lack of clotting factors prevents fibrin formation, and fibrin formation is necessary to maintain blood clots. Hemophilia patients will not bleed more than people without hemophilia, but will continue to bleed for a longer period of time. In severe hemophilia patients, even minor injuries can result in blood loss for a few days or weeks, or even complete healing. In areas such as the brain or joints, this can be fatal or permanently debilitating.

Other bleeding disorders that can be treated with the antibodies of the invention include acquired platelet function defects, congenital platelet function defects, congenital protein C or S deficiency, disseminated intravascular coagulation (DIC), factor II deficiency, factor V deficiency, Factor VII deficiency, factor X deficiency, factor XII deficiency, idiopathic thrombocytopenic purpura (ITP), and Wilberg's disease.

B. Pharmaceutical compositions, routes and dosages

Pharmaceutical compositions comprising one or more anti-TFPI prodrug antibodies can be administered to a patient alone or in combination with other agents, drugs or clotting factors to treat hemophilia or other coagulopathy. A "therapeutically effective dose" of an anti-TFPI prodrug antibody means that the anti-TFPI prodrug antibody promotes coagulation or reduces the amount of bleeding time. Determination of a therapeutically effective dose is within the skill of the artisan.

The therapeutically effective dose can be estimated initially in cell culture assays or in animal models (usually rats, mice, rabbits, dogs or pigs). Animal models can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine the available dose and route when administered in a human body.

Therapeutic efficacy and toxicity, eg ED anti-TFPI prodrug antibody ₅₀ (the dose therapeutically effective in 50% of the population) and the LD ₅₀ (resulting dose lethal to 50% of the population) can be through standard pharmaceutical procedures in cell cultures or experimental animals, Determined in. The dose ratio of toxicity to therapeutic effect is the therapeutic index and it can be expressed as the ratio, LD ₅₀ /ED ₅₀ .

Pharmaceutical compositions exhibiting a large therapeutic index are preferred. Data obtained from cell culture assays and animal studies were used to formulate a range of doses for human use. Doses contained in the composition is preferably within a range of circulating concentrations that include an ED ₅₀ with little or no toxicity. The dosage is based on the dosage form employed, and the patient's sensitivity and route of administration vary within this range.

The precise dose will be determined by the clinician based on factors associated with the patient in need of treatment. The dosage and administration are adjusted to provide a sufficient amount of anti-TFPI prodrug antibody or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, the general health of the individual, the age, weight and sex of the individual, the diet, the time and frequency of administration, the combination of drugs, the sensitivity of the response, and the tolerance/response to the treatment. . The long acting pharmaceutical composition can be administered every 3 to 4 days, every week, or every two weeks, depending on the half-life and clearance rate of the particular formulation.

In some embodiments, the in vivo therapeutically effective dose of the anti-TFPI prodrug antibody is in the range of from about 5 [mu]g to about 100 mg/kg, from about 1 mg to about 50 mg/kg, from about 10 mg to about 50 mg/kg of patient body weight.

The mode of administration of a pharmaceutical composition comprising an anti-TFPI prodrug antibody can be any suitable route for delivery of the antibody to the host (e.g., subcutaneous, intramuscular, intravenous or intranasal administration).

In some embodiments, the anti-TFPI prodrug antibody is administered without additional therapeutic agents. In some embodiments, the anti-TFPI prodrug antibody is administered in combination with other agents, such as drugs or clotting factors, to increase the initial production of thrombin while ensuring that thrombin levels are kept below It may cause a range of thrombosis in some people with coagulopathy. The anti-TFPI prodrug antibody can be administered before, after, or at substantially the same time as other agents are administered.

Instance

The following examples are included to further illustrate various aspects of the invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques and/or compositions that the inventors have found to be effective in the practice of the particular embodiments and are therefore considered to be Preferred mode. However, it is to be understood by those skilled in the art that the invention may be <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

Example 1 - Anti-TRPI Prodrug Antibody Design Strategy

At least three strategies have been devised to mask anti-TFPI activity, but other strategies are expected. An anti-tissue factor antibody domain, an anti-erythrocyte antibody domain or an albumin binding peptide can be used as a masking domain. The masking function can involve binding of the prodrug antibody to a first target (such as tissue factor, red blood cells or albumin). When the anti-TFPI prodrug antibody is in its potential form, there is no significant reduction in binding or binding to TFPI until the masking region is cleaved by a protease produced by the coagulation cascade, as shown in FIG. Different forms of prodrug antibodies have been designed. While all forms include the Fc region (which allows for FcRn binding to provide a longer half-life), the variable regions can be modified, including tandemly linked variable regions, scFv-variable region fusions, peptide variable region fusions, and the like. Representative images of these prodrug antibodies are shown in Figure 2. The parent antibody to the prodrug antibody currently envisioned is found in the human antibody library. These antibodies have been extensively optimized to increase their affinity and functionality. The parental antibodies, gA200 and gB9.7, have high affinity and specificity for human TFPI and promote tissue factor (TF) to initiate coagulation.

Masking methodology A. Tissue factor binding

Tissue factor (TF) is a protein present in the subendothelial tissue and white blood cells and is necessary for eliciting thrombin formation from the zymogen prothrombin. Tissue factor is exposed to the bloodstream only when damage occurs to initiate coagulation. Thus, targeting TF allows activation of anti-TFPI prodrug antibodies at the site of injury. The TF binding masking domain incorporated into the anti-TFPI prodrug antibody can be a TF binding antibody, peptide or another scaffold that does not block TF function.

B. Anti-erythrocyte binding

RBC (red blood cells) has been used as a carrier or reservoir for the delivery of drugs or enzymes. RBC is biocompatible, biodegradable, has a long circulating half-life, and can be loaded with a variety of biologically active substances. Surface modification using antibodies has been shown to improve their target specificity and increase their circulating half-life. In the present invention, an anti-RBC antibody is used as a masking domain fused to the N-terminus of an anti-TFPI antibody. This anti-TFPI prodrug antibody produces a prodrug that has a longer latency than the unmodified parental anti-TFPI antibody. The binding of the prodrug to RBC will further reduce its ability to bind TFPI until the masking domain is cleaved.

C. Albumin binding peptide

Albumin has become a universal carrier for therapeutic and diagnostic agents, primarily for the diagnosis and treatment of diabetes, cancer, rheumatoid arthritis and infectious diseases. Human serum albumin is the most abundant protein in the body and has a concentration of about 40 mg/mL in the circulation. Albumin has a molecular weight of 67 kDa. The albumin binding moiety can be used as a masking domain fused to the N-terminus of the anti-TFPI antibody, resulting in an anti-TFPI prodrug antibody having a longer latency than the parental anti-TFPI antibody. While albumin-binding peptides are used in prodrug constructs, the albumin binding moiety can be a peptide, a native albumin binding domain, a scaffold, an antibody, or an antibody fragment (such as a Fab, scFv, domain antibody, and other derivatives).

D. Protease screening and design of cleavage sites

When damage occurs, tissue factor (TF) becomes exposed to the bloodstream and activates Factor VII to form a TF/FVIIa complex. The TF/FVIIa complex thereby activates factor X and FXa, which activates prothrombin to thrombin, resulting in fibrin formation and blood coagulation. The primary role of the tissue factor pathway is to produce a "thrombin burst" in which thrombin (a most important component of the coagulation cascade due to its feedback activation) is instantaneously released. In addition, a range of other coagulation factors are activated in the coagulation cascade:

̇TF-FVIIa activates FIX and FX.

̇FVII itself is activated by thrombin, FXIa, FXII and FXa.

̇FXa and its cofactor FVa form a prothrombinase complex that activates prothrombin to thrombin.

Thallium thrombin then activates other components of the coagulation cascade, including FV and FVIII, which activate FVIII release that binds to vWF. Finally, thrombin activates FXI, which in turn activates FIX.

̇FVIIIa is a cofactor for FIXa, and together they form a "tenase" complex that activates FX; and thus continues to circulate. ("X enzyme" is an abbreviation for the "ten" and suffix "-enzyme" of the enzyme).

Many of the aforementioned proteases, such as FVIIa, FXa, and thrombin, can be used to activate anti-TFPI prodrug antibodies. In the present invention, the cleavage sites of FXa and thrombin are designed as masking domains, but it is envisioned that any of the other proteases mentioned may also be incorporated into the anti-TFPI prodrug antibody.

Example 2. Vector map of prodrug antibodies

Nine gA200-prodrug heavy chain (HC) and six gA200 light chain variants (LC) were constructed using Infusion cloning (Clontech) into the expression plasmid pTTF5. All variants contained the six amino acid cleavage sites EGRTAT of FXa. Mutant proteins contain various N-terminal and C-terminal deletions on either side of the cleavage site. Representative profiling maps of the HC1 mutein and the LC1 mutein are shown in Figures 3A-B. The DNA sequences of the 15 light and heavy chain variants are shown in Figure 12, respectively. The HC muteins were named HC1 to HC9, and the LC muteins were named LC1 to LC6. A representative vector map of scFV (anti-RBC or anti-TF) fused to anti-TFPI is shown in Figures 4A-4C, and a representative vector map of the albumin-binding peptide fused to anti-TFPI is shown in Figure 5A-B. in. Figures 13-14 provide sequences comprising constructs of various light and heavy chain combinations with engineered cleavage sites.

Example 3 - Performance and Production of Prodrug Antibodies

When Chinese hamster ovary cells were used as host cells, Amaxa's nuclear transfection (Neucleofection) technique was used for transfection. Briefly, 2 x 10 ⁶ cells per reaction were centrifuged at 1000 rpm for 5 minutes into pellets. The cell pellet was resuspended in 100 μl of Nucleofector solution V per reaction. 2 μg of pQM1-3E10sc-gA200HC and pQM1-gA200LC, pQM1-Ter119sc-gA200HC and pQM1-gA200LC, or pQM1-56E4-gA200HC and pQM1-56E4-gA200LC, respectively, were added to the cells. The DNA/cells in solution V were then transferred to a Nucleocuvette container. Electroporation was performed using the program U024 in Nucleofector®. Immediately after electroporation, 0.5 mL of warm medium was added to the cells, and then transferred to a 6-well plate containing 4.5 mL of pre-warmed Qmix 1 medium (without antibiotics) per well and placed back on a shaker at 37 ° C incubator. The performance of the prodrug antibody was determined 3-4 days after transfection. For positively expressing cells, a stable pool is generated. The cells were diluted to 0.5 x 10 ⁶ /mL and G418 was added up to 0.7 mg/mL. Once the cell density reached 3-4 x 10 ⁶ /mL, the cells were again diluted to 0.4 x 10 ⁶ /mL and maintained in Qmix ¹ containing 0.7 mg/mL of G418. After about two weeks of screening, it is the production phase. When the cell viability was restored to >95% and the cell density reached 3.5-4 x 10 ⁶ /mL, the culture temperature was switched to 30 °C. Conditioned medium was collected 4-7 days after temperature conversion. Cells were removed by centrifugation at 5000 rpm for 30 minutes. The conditioned medium was concentrated 5 times using a Millipore concentrator, followed by additional centrifugation at 9000 rpm for 40 minutes.

When HEK293-6E cells were used as host cells, they were maintained as suspension cultures in F17 medium supplemented with 4 mM L-glutamic acid, 0.1% Pluronic F68, and 25 mg/L G418. Transfection was carried out using polyethyleneimine (PEI, 25 KD, linear). Briefly, 1 x 10 ⁶ cells/ml was inoculated one day prior to transfection. On the day of transfection, the cell density was adjusted to 1.7 x 10 ⁶ /ml. For transfection of 1 L cells, 0.5 mg of each VEC-4581 and VEC-4568 (for TPP2652), or VEC-4583 and 4568 (for TPP2654) were diluted in 500 ml of F17 medium, while 2 ml of PEI (PEI stock solution) It was diluted to 500 ml of F17 at 1 mg/ml. The diluted DNA and PEI were pooled and added to the cells after 10' incubation at room temperature. The cells were then returned to a 125 rpm shaker in a 37 °C incubator. Twenty four hours after transfection, cells were fed with 1% ultra low IgG FBS and 0.5 mM valproic acid. The performance of the prodrug antibody was measured 3-4 days after transfection, and the performance was terminated when the cell viability was reduced to 70%. The conditioned medium was removed by centrifugation at 2000 rpm for ten minutes to remove the cells, followed by additional centrifugation at 9000 rpm for 40 minutes.

Example 4 - Purification of anti-TFPI prodrug antibodies

Prodrug proteins were purified from CHO cell conditioned medium using a MabSelect Protein A column (5 mL HiTrap, GE HealthCare, #28-4082-55). The medium is concentrated 5 to 10 times by ultrafiltration or used without concentration. Before pumping the medium to the column at a flow rate of 1-1.5 mL/min, the column was "equilibrated buffer" (50 mM Tris-HCl, 150 mM NaCl, pH 7.0). Balance. After loading, the column was washed with 5 to 10 column volumes (CV) of equilibration buffer at a flow rate of 4 mL/min. It was then re-equilibrated with "Acetate Wash Buffer" (50 mM sodium acetate, 150 mM NaCl, pH 5.4).

The bound protein was eluted from the column using a three-step dissolving at a flow rate of 1 mL/min: (1) 50 mM sodium acetate, 150 mM NaCl, pH 3.4; (2) 50 mM sodium acetate, 150 mM NaCl, pH 3.2; and (3) 100 mM glycine-HCl, pH 3.0. The fraction (1 mL/dissolved) was collected into a vial containing 1 ml of "mixing buffer" (50 mM sodium acetate, 50 mM NaCl, pH 5.4) to increase the pH. With 100mM glycine, pH 2.8 regeneration column, and then washed with ₂ O dH and stored in 20% ethanol.

The fractions containing the protein (as determined by absorbance monitoring at 280 nm) were pooled and the buffer was exchanged into the formulation buffer by overnight dialysis at 4 °C or by rotary desalting column. The final concentration of the protein solution was achieved by ultrafiltration using a 10 kDa concentrator. Any precipitate that may form during concentration or dialysis is removed by centrifugation at 2000 xg for 30 minutes. The final sample was sterile filtered using 0.22 mm 匣.

The purified protein was characterized by SDS-PAGE, analytical size screening chromatography (aSEC), and Western blotting. Endotoxin levels were also measured. The purity according to aSEC and SDS-PAGE is usually greater than 90%. SDS-PAGE is shown in Figure 6.

Example 5-GA200 and 56E4-GA200 in vitro TFPI binding ELISA

Maxisorb 96-well plates (Nunc) were coated with 1 μg/mL TFPI in PBS overnight at 4 °C. The plate was blocked in 5% non-fat dry milk PBS/0.5% Tween-20 for 1 hour at room temperature. Serial 3-fold dilutions of undigested and digested antibodies were added to each well (100 [mu]L/well) and incubated for 1 hour at room temperature. The plate was washed 5 times with PBS-T. A secondary anti-Fab-HRP binding antibody (100 μL of 1:10,000 dilution) and Amplex Red (Invitrogen) solution were added for detection. The HSA-bound prodrug antibody slightly reduced binding to TFPI compared to its parent anti-TFPI antibody gA200, as seen in Figure 7.

Example 6 - RBC Binding ELISA of TER119SC-GA200 Prodrug Antibody

ELISA was used to test the binding of prodrug antibodies to RBC. 100 μL of murine RBC (without Ca or Mg) resuspended in DPBS was applied to the wells of a clear 96-well Maxisorp microtiter plate at a concentration of 10 ⁷ /mL. The plate was sealed with tape and incubated overnight at 4 °C. Wells were washed once with DPBST (DPBS + 0.05% Tween 20) and then blocked with 5% milk/DPBST for 1 hour at room temperature. The blocking buffer was discarded and 50 μL of the diluted sample was added to each well. Samples were serially diluted 1:3 in PBS. The plates were incubated for 1 hour at room temperature and then quickly washed 5x with DPBST. 100 μL of secondary antibody (HRP-goat anti-hFAB, Jackson ImmunoResearch, cat # 109-035-097) diluted 1:5000 in PBST was added to each well. The plates were incubated for 1 hour at room temperature and then washed 5X with DPBST. HRP substrate (Amplex Red, Invitrogen A22177) was added and fluorescence readings were taken on an SpectraMax M2e (Molecular Devices) at an excitation wavelength of 485 nM and an emission wavelength of 595 nM. The concentration of prodrug antibody bound to RBC can be seen in Figure 8 over 10 nM.

Examples of anti-TFPI 7- BIACORE ^TM Analysis prodrug antibody

Method. Human TFPI was immobilized on a CM4 or CM5 wafer using an amine coupling kit according to the manufacturer's instructions. Anti-TFPI prodrug antibodies or parental anti-TFPI antibodies were passed through the system with or without 15 [mu]g/mL human serum albumin (HSA) at 10 [mu]g/mL antibody. The binding level was measured in 2 seconds after each injection was completed. In the kinetic analysis, antibodies were injected at a range of concentrations followed by a 30-resolution time. The dissociation and association rates of antibodies were modeled using the BiaEvaluation software.

Results. Figure 9 shows different prodrug antibodies that bind to human TFPI. As shown in Figure 9, in the absence of HSA, the ABP-gA200 prodrug antibody binds to TFPI in 179 reflectance units (RU), while in the presence of HSA, the signal is reduced by 80% up to 36 RU. In contrast, human albumin did not significantly affect the binding of the parent antibody gA200 to TFPI.

The RBC binding prodrug antibody Ter119scFv-gA200 binds to TFPI at the same level as gA200, while the TF-bound prodrug antibody 3E10 scFv-gA200 binds to TFPI at a residual level of 22%.

To further measure the binding of their prodrug antibodies, a kinetic analysis was performed to measure the affinity. As shown in Table 1a, the anti-TF prodrug antibody 3E10scFv-gA200 and the anti-RBC prodrug antibody Ter119scFv-gA200 reduced binding to TFPI by 29.71 fold and 14.66 fold, respectively. The albumin-binding prodrug antibody ABP-gA200 did not reduce binding to TFPI, but its binding to TFPI was also reduced by 15.34-fold after mixing with HSA and culturing.

To further optimize cleavage of ABP-gA200, the cleavage site of ABP-gA200 was modified. Different cleavage sites are inserted between the albumin binding peptide and the heavy chain variable region. As shown in Figures 14a-b, the spacer GGGGS is inserted around the cleavage site. While TPP-2651, TPP-2652, TPP-2653, and TPP2655 contain a thrombin cleavage site, TPP-2654 contains a linker that can be cleaved by FXa and thrombin. As shown in Table 1b, in the presence of human or monkey albumin, these antibodies reduced their binding to TFPI by up to 28.6-fold (TPP-2654) and 39.6-fold (TPP-2652), while the parent antibody gA200 was Albumin has similar TFPI binding affinity in the absence or presence of albumin. Data for TPP-2653 is not displayed. The prodrug antibody is purified and digested by protease (whether thrombin or factor Xa). After removal of these proteases, the antibodies were analyzed using LC-MS. The results indicated that the protease cleaves the albumin binding peptide. Representative data for TPP-2652 and TPP-2654 are shown in Figures 16a-C.

To further balance the cleavage efficiency of the anti-TFPI prodrug and the masking function, we altered the linker length and truncated the FR1 domain of antibody gA200. The amino terminal sequence of the prodrug with ABP is shown in Figure 14.

Example 8 - Analysis of thrombin generation by anti-TFPI prodrug antibody

Thrombin generation assay (TGA) of TFPI prodrug antibodies was performed using human HemA plasma. The platelet-poor plasma (PPP) reagent and calibrator were reduced in 1 mL of distilled water. A 1:2 serial dilution of the anti-TFPI prodrug antibody (from a final concentration of 100 nM to 1.56 nM) was added to HemA human plasma. Only plasma samples were used as controls. In a 96-well TGA pan, 20 μL of PPP reagent or calibrator was added to each well, followed by 80 μL of plasma samples containing different concentrations of anti-TFPI prodrug antibodies. The tray is placed in the TGA instrument, and the instrument automatically assigned FluCa 20μL of each well (by mass Fluo + CaCl _2). Thrombin generation was measured for 60 minutes. When Ter119scFv-gA200 was tested in TGA, mouse RBC ghosts (RBC ghost) were incorporated into HemA plasma. Ter119scFv-gA200 was incubated with RBC-GOLD for 15 minutes at room temperature.

As shown in Figures 10A-B, 56E4-gA200 produced a thrombin peak that was lower than its parent antibody gA200, indicating that human albumin in plasma may reduce anti-TFPI activity. The thrombin shape produced by 56E4gA200 is low and the broad peak also indicates that the antibody is less active at time zero and may become activated by the FXa or thrombin produced.

The feasibility of converting a prodrug TFPI antibody to a more active TFPI antibody under physiological conditions is established by: 1) the ability of exogenous thrombin (physiological level) to increase the TGA response of the TFPI Ab vector, and 2 FXa and thrombin are produced in the TGA reaction and monitored for subsequent increase in the response caused by the TFPI antibody.

At physiologically achievable levels, exogenous thrombin addition directly assesses the susceptibility of the prodrug TFPI antibody to the enzyme and pre-incubated with 1200 nM Ab with 0.5 to 2.5 U/mL thrombin for 1 hour, followed by The thrombin was not activated for 1 hour using 0.5 to 2.5 U/mL thrombin-specific irreversible inhibitor hirudin. To measure the effect of hirudin residues in the TGA reaction, buffer was used instead of thrombin to assess the effect of hirudin residues on the TGA response. In some cases, an unrelated Ab or parental antibody gA200 (no albumin masking or protease sensitive site) was used in place of the original TFPI Ab as a control. The antibody-thrombin- hirudin mixture was serially diluted to an Ab concentration of 10 to 100 nM, and the mixture was additionally diluted 1:10 in a TGA reaction. The TGA reaction was carried out as described above, except that the initiator used was PPP-Low, which contained 1 pM TF-4 μM platelets. TGA results using prodrug TFPI antibodies were subtracted from TGA results using incoherent antibodies.

The TGA profile of pre-TFPI Ab TPP-2654 before and after protease cleavage is shown in Figure 10C. The TGA response of the parental gA200 was not altered by thrombin incubation, while TPP-2654 (which contains thrombin and FXa sensitive cleavage sites) showed increased response when pre-incubated with thrombin. However, the peak TGA response is less than the use of gA200, indicating that complete disclosure of TFPI Ab activity may require higher levels of thrombin present or optimized protease sensitive sites to increase the efficiency of protease cleavage.

Concentration titration experiments using a range of thrombin concentrations (0.5 to 2.5 U/mL) have determined that the conversion of prodrug TFPI antibodies to more active TFPI antibodies can be achieved at 1 U/mL thrombin, in vivo. The achievable level (Fig. 10D).

To assess whether FXa or thrombin can more efficiently convert prodrug antibodies to active TFPI antibodies, particularly in prodrug antibodies containing protease sensitive sites, assess the effect of increasing FXa and thrombin in situ. In situ generation of FXa is increased by increasing the concentration of TF used as an initiator. In these experiments, the TF concentration was changed from 1 pM to 5 pM in the TGA reaction by using PPP-Low (1 pM TF-4 μM PL) or PPP reagent (5 pM TF-4 μM PL) as an initiator. Increasing TF will increase FXa through the direct action of TF-FVIIa and then increase thrombin generation. The TGA reaction was carried out as described above, and the results were analyzed by comparing the difference in reaction between the TGA reactions using 1 pM and 5 pM TF.

The FXa and thrombin susceptibility of TPP-2654 showed a significant increase in the peak thrombin response (Δ peak) between the 1 pM and 5 pM TF initiators (Fig. 10E), especially the prodrug TFPI antibody was pre-extended with 2.5 U/mL. In the case of cultivation. The ability of the pre-TFPI Ab TPP-2654 to increase the peak thrombin response exceeds the response achieved by the initial thrombin pre-incubation, indicating that FXa cleavage further promotes complete disclosure of the albumin-binding peptide.

The TGA response of the original TFPI Ab (TPP-2652), in which the albumin-binding peptide was masked for removal by thrombin cleavage, is shown in Figures 10F and 10G. The thrombin sensitivity of TPP-2652 is shown in Figure 10F, indicating that only a small increase in TGA response was detected at the highest level of test antibody at the maximum concentration of test thrombin (2.5 U/mL). FXa (and thrombin) increased by increasing the TF concentration used to initiate TGA only slightly increased the TGA response of TPP-2652 (Fig. 10F). This is in contrast to a significant increase in the thrombin response of TPP-2654 when TDP-2654 pretreated with thrombin was further exposed to FXa produced using 5 pM TF (compare Figure 10E with Figure 10G).

These results suggest that different protease sensitive sites may affect the conversion of prodrug TFPI antibodies to active TFPI Ab.

Example 9 - FXA Analysis of Anti-TFPI Prodrug Antibodies Materials and Methods

The following reagents were used for FXa analysis:

Assay buffer: 1X buffer was 25 mM Hepes 7.4, 100 mM NaCl, 5 mM CaCl ₂ , 0.1% BSA.

TFPI-R&D (Cat# 2974-PI, MW~35kDa). TFPI was reduced to 100 μg/mL (2.86 μM) by adding 10 μL of 25 mM Tris and 150 mM NaCl, pH 7.5 according to the product insert instructions. The 2.86 μM stock solution was diluted 1/143 to produce a 20 nM working stock solution.

FXa-Haematologic Technologies (Cat# HCX-0060, MW-58.9 kDa) was pre-formed in assay buffer in 2 μM aliquots and stored at -80 °C. A 2 μM stock solution was diluted 1/1000 as a 2 nM working stock solution.

S-2765-Chromogenix (Cat # S-2765, MW-714.6Da) produced a 5 mM working stock solution by dissolving 25 mg of lyophilized material in 7 mL of distilled water. A 5 mM working stock was added directly to the assay wells.

A 4X dose curve for anti-TFPI antibodies was generated in assay buffer. 60 μL of each antibody was combined with a 4X (20 nM) concentration of TFPI. The antibody/TFPI mixture was incubated for 30 minutes at room temperature. 120 μL of 2X (2 nM) concentration of FXa was added to the Ab/TFPI mixture and incubated at room temperature for 30 minutes. The Ab/TFPI/FXa mixture was then transferred to the assay plate in 100 [mu]L per well in a two-fold format followed by 20 [mu]L of 5 mM substrate. The disk was read in a dynamic manner at 405 nm for 3 minutes in a Molecular Devices Spectramax disk reader. When tested with albumin-conjugated anti-TFPI prodrug antibody 56E4-gA200, 34 [mu]L of 8X anti-TFPI antibody combined different concentrations of 34 [mu]L albumin in 96 well round bottom polypropylene disks. The solution was then incubated at room temperature for 15 minutes and 68 μL of 20 nM (4X) TFPI was added to α-TFPI Ab/albumin.

As shown in FIG. 11, gA200 V _max is not subject to influence human albumin or monkey. Albumin binding antibody 56E4-gA200 prodrug albumin concentration decreased with increasing V _max. When the human or monkey albumin concentration reached 5 μM, the anti-TFPI activity of the prodrug antibody was inhibited by 90%.

Example 10 - Protease digestion of anti-TFPI prodrug antibodies

Protease cleavage of the prodrug and protease depletion were performed using the Novagen thrombin cleavage capture kit (69022-3) and the Novagen Factor Xa kit (69037-3).

Biotinylated thrombin was used for prodrug cleavage as outlined below.

The 50 uL reaction of each prodrug contained 5 μg of prodrug, 5 μL of 10 x set of thrombin cleavage/capture buffer, 1 unit of thrombin, and 50 μL of deionized water. Incubate the reaction calendar at 37 ° C 1 hour. After the cleavage reaction, biotinylated thrombin was removed as a ratio of 16 ml of static resin per unit of enzyme (32 ml of 50% syrup) to avidin agarose (provided in the kit). After the agarose was added to the reaction tube, the tube was incubated at room temperature for 30 minutes and gently shaken. The entire reaction was transferred to the sample cup and spin filter provided by the kit. The tube was then centrifuged at 500 xg for 5 minutes. The filtrate in the collection tube contains the cleaved prodrug, without biotinylated thrombin.

When Factor Xa was used for prodrug cleavage, 50 μL of each prodrug contained 5 μg of prodrug, 5 μL of 10x kit of Factor Xa cleavage/capture buffer, 1 unit of Factor Xa, and deionized water to 50 μL of total volume. The reaction was incubated at 37 ° C for 1 hour. After the cleavage reaction, to Xarrest ^TM agarose (provided within the kit) to remove Factor Xa. Xarrest agarose was first equilibrated by adding 11 volumes of 1x Factor Xa cutting/capture buffer to each resting resin volume of Xarrest agarose. Xarrest agarose was centrifuged at 1000 xg for 5 minutes. Remove the supernatant and discard. The agarose was resuspended in 10 volumes of 1X Factor Xa cleavage/capture buffer and centrifuged at 1000 xg for 5 minutes. Remove the supernatant and discard. A static resin volume of 1X Xa factor cutting/capture buffer was added to the tube and the resin was fully resuspended. The prepared Xarrest agarose was transferred to a sample cup (contained in the kit) with a 2 ml spin filter. The total volume of the prodrug cleavage reaction was added to the prepared Xarrest agarose. The tube was incubated at room temperature for 5 minutes and centrifuged at 1000 xg for 5 minutes to remove the Xarrest agarose. The bound factor Xa is retained in the sample cup and the cleaved prodrug flows into the filtrate tube during centrifugation.

Example 11 - LC-MS of anti-TRPI prodrug antibodies

LC-MS analysis of anti-TFPI prodrug antibodies TPP-2652 and TPP-2654 was performed under intact or reducing conditions. For intact proteins, 2 μg was loaded directly onto the PLRP column. Regarding the reduction of the sample, the test sample was treated with 10 mM DTT at 37 ° C for 30 minutes before loading onto the PLRP column.

LC separation was performed using an Agilent 1200 Capillary LC System with PLRP-S (8 μm 4000A, 0.3 x 150 mm) at 70 °C. The buffer system for LC was A: water plus 0.1% formic acid + 0.01% TFA, B: acetonitrile plus 0.1% formic acid + 0.01% TFA, flow rate 10 μL/min. Gradient: 10% B within 2 minutes, 90% B within 15 minutes, 90% B for 5 minutes, 10% B balance for 10 minutes.

MS analysis was performed using an Agilent 6520 Q-TOF system. The conditions were DualEsi source, gas temperature: 350 ° C, dry gas: 7 psi, nebulizer: 10 psi, scanning range: 500-3000 amu, 1 spectrum / s. Two experiments per cycle: reduction form of 3500v, 175v cutting voltage, 65v cone voltage, and intact protein of 4000v, 350v cutting voltage, 100v cone voltage. Reference ions: 1221.990637 and 2421.91399 amu, 50 ppm window, Min 1000 purified prodrug antibody and digested with protease (thrombin or factor Xa). After removal of these proteases, the antibodies were analyzed using LC-MS. The results indicated that the protease cleaves the albumin binding peptide. Representative data for TPP-2652 and TPP-2654 are shown in Figures 16a-c.

All of the compositions and methods disclosed and claimed herein can be made and carried out in accordance with the present disclosure without undue experimentation. Although the compositions and methods of the present invention have illustrated preferred embodiments, various variations may be applied to the compositions and methods, as well as to the steps or sequence of steps of the methods described herein, without Without departing from the spirit, spirit and scope of the invention. More specifically, certain chemically and physiologically relevant agents are apparently capable of replacing the agents described herein to achieve the same or similar results. All such similar substitutes and modifications are obvious to those skilled in the art, which are within the spirit, scope and concept of the invention as defined by the appended claims.

<110> Bayer Health Care Co., Ltd.

<120> Anti-tissue factor pathway inhibitor prodrug antibody PRO-DRUG ANTIBODIES AGAINST TISSUE FACTOR PATHWAY INHIBITOR

<130> BAYR.P0004WO

<150> 61/794,024

<151> 2013-03-15

<160> 256

<170> PatentIn version 3.5

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<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 95

<210> 96

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 96

<210> 97

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 97

<210> 98

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 98

<210> 99

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 99

<210> 100

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 100

<210> 101

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 101

<210> 102

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 102

<210> 103

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 103

<210> 104

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 104

<210> 105

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 105

<210> 106

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 106

<210> 107

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 107

<210> 108

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 108

<210> 109

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 109

<210> 110

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 110

<210> 111

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 111

<210> 112

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 112

<210> 113

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 113

<210> 114

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 114

<210> 115

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 115

<210> 116

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 116

<210> 117

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 117

<210> 118

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 118

<210> 119

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 119

<210> 120

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 120

<210> 121

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 121

<210> 122

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 122

<210> 123

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 123

<210> 124

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 124

<210> 125

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 125

<210> 126

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 126

<210> 127

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 127

<210> 128

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 128

<210> 129

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 129

<210> 130

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 130

<210> 131

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> Fab-1 light chain

<400> 131

<210> 132

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage sites and linkers

<400> 132

<210> 133

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> Fab-1 heavy chain

<400> 133

<210> 134

<211> 1590

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence encoding Fab-1

<400> 134

<210> 135

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> Fab-2 light chain

<400> 135

<210> 136

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<223> Fab-2 heavy chain

<400> 136

<210> 137

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Protease cleavage site

<400> 137

<210> 138

<211> 1530

<212> DNA

<213> Artificial sequence

<220>

<223> Fab-2 coding sequence

<400> 138

<210> 139

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> IgG2-linker 1, light chain amino acid sequence

<400> 139

<210> 140

<211> 458

<212> PRT

<213> Artificial sequence

<220>

<223> IgG2-linker 1, heavy chain amino acid sequence

<400> 140

<210> 141

<211> 684

<212> DNA

<213> Artificial sequence

<220>

<223> IgG2-linker 1, light chain DNA sequence

<400> 141

<210> 142

<211> 1374

<212> DNA

<213> Artificial sequence

<220>

<223> IgG2-linker 1, heavy chain DNA sequence

<400> 142

<210> 143

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> IgG2-linker 2, light chain amino acid sequence

<400> 143

<210> 144

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> IgG2-linker 2, heavy chain amino acid sequence

<400> 144

<210> 145

<211> 654

<212> DNA

<213> Artificial sequence

<220>

<223> IgG2-linker 2, light chain DNA sequence

<400> 145

<210> 146

<211> 1344

<212> DNA

<213> Artificial sequence

<220>

<223> IgG2-linker 2, heavy chain DNA sequence

<400> 146

<210> 147

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Thrombin cleavage site

<220>

<221> variant

<222> (1)..(2)

<223> Xaa=hydrophobic amino acid

<220>

<221> variant

<222> (5)..(6)

<223> Xaa = non-acidic amino acid

<400> 147

<210> 148

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Factor Xa cleavage site

<220>

<221> variant

<222> (2)..(2)

<223> Xaa=Glu or Asp

<400> 148

<210> 149

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 149

<210> 150

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid 95-152 of SEQ ID NO: 133

<400> 150

<210> 151

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 151

<210> 152

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 152

<210> 153

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 153

<210> 154

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 154

<210> 155

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 155

<210> 156

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 156

<210> 157

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 157

<210> 158

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 158

<210> 159

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 159

<210> 160

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 160

<210> 161

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 161

<210> 162

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 162

<210> 163

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 163

<210> 164

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 164

<210> 165

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 165

<210> 166

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 166

<210> 167

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 167

<210> 168

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 168

<210> 169

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 169

<210> 170

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 170

<210> 171

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 171

<210> 172

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 172

<210> 173

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 173

<210> 174

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 174

<210> 175

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 175

<210> 176

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 176

<210> 177

<211> 442

<212> PRT

<213> Homo sapiens

<400> 177

<210> 178

<211> 212

<212> PRT

<213> Homo sapiens

<400> 178

<210> 179

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<223> Protease can cleave anti-TFPI scFv antibody

<400> 179

<210> 180

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<223> Protease cleavage anti-TFPI scFv antibody coding sequence

<400> 180

<210> 181

<211> 569

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 181

<210> 182

<211> 565

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 182

<210> 183

<211> 561

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 183

<210> 184

<211> 566

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 184

<210> 185

<211> 562

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 185

<210> 186

<211> 558

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 186

<210> 187

<211> 563

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 187

<210> 188

<211> 559

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 188

<210> 189

<211> 555

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 189

<210> 190

<211> 331

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 190

<210> 191

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 191

<210> 192

<211> 323

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 192

<210> 193

<211> 329

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 193

<210> 194

<211> 325

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 194

<210> 195

<211> 321

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 195

<210> 196

<211> 690

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 196

<210> 197

<211> 683

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 197

<210> 198

<211> 212

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 198

<210> 199

<211> 463

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 199

<210> 200

<211> 705

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 200

<210> 201

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 201

<210> 202

<211> 699

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 202

<210> 203

<211> 700

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 203

<210> 204

<211> 717

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 204

<210> 205

<211> 705

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 205

<210> 206

<211> 710

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 206

<210> 207

<211> 578

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 207

<210> 208

<211> 334

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 208

<210> 209

<211> 565

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 209

<210> 210

<211> 322

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 210

<210> 211

<211> 568

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 211

<210> 212

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 212

<210> 213

<211> 482

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 213

<210> 214

<211> 476

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 214

<210> 215

<211> 494

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 215

<210> 216

<211> 482

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 216

<210> 217

<211> 472

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 217

<210> 218

<211> 463

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 218

<210> 219

<211> 482

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 219

<210> 220

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 220

<210> 221

<211> 476

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 221

<210> 222

<211> 494

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 222

<210> 223

<211> 482

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 223

<210> 224

<211> 472

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 224

<210> 225

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 225

<210> 226

<211> 476

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 226

<210> 227

<211> 473

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 227

<210> 228

<211> 472

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 228

<210> 229

<211> 470

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 229

<210> 230

<211> 468

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 230

<210> 231

<211> 468

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 231

<210> 232

<211> 482

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 232

<210> 233

<211> 479

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 233

<210> 234

<211> 442

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 234

<210> 235

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 235

<210> 236

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 236

<210> 237

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 237

<210> 238

<211> 54

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 238

<210> 239

<211> 63

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 239

<210> 240

<211> 63

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 240

<210> 241

<211> 82

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 241

<210> 242

<211> 69

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 242

<210> 243

<211> 63

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 243

<210> 244

<211> 73

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 244

<210> 245

<211> 67

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 245

<210> 246

<211> 64

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 246

<210> 247

<211> 63

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 247

<210> 248

<211> 61

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 248

<210> 249

<211> 59

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 249

<210> 250

<211> 59

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 250

<210> 251

<211> 73

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 251

<210> 252

<211> 70

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 252

<210> 253

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 253

<210> 254

<211> 64

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 254

<210> 255

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 255

<210> 256

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 256

Claims (35)

An antibody comprising: (a) a first variable domain comprising a first light chain and a first heavy chain variable region, the first variable domain binding to a tissue factor pathway inhibitor (TFPI); (b) linked to a masking domain of the first light chain and/or the amine-terminal end of the first heavy chain variable region; and (c) a protease cleavable linker inserted into the first light chain and/or the first heavy chain variable region And between the masking domain. The antibody of claim 1, wherein the protease cleavable domain is a thrombin, plasmin, factor VIIa or factor Xa cleavage site. The antibody of claim 1, wherein the masking domain comprises a second variable domain comprising a second light chain and a second heavy chain variable region. The antibody of claim 1, wherein the antibody is IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE antibodies. The antibody of claim 1, wherein the antibody is a human or a humanized antibody. The antibody of claim 1, wherein the antibody is a single chain antibody. The antibody of claim 1, wherein the antibody is bivalent and comprises two masking domains, one linked to the amine terminus of each of the first light chain variable regions. The antibody of claim 1, wherein the antibody is bivalent and comprises two masking domains, one linked to the amine terminus of each of the first heavy chain variable regions. The antibody of claim 1, wherein the antibody is bivalent and comprises four masking domains, one linked to each of the first light chain variable region and the amine terminal of each of the first heavy chain variable regions. The antibody of claim 9, wherein the two masking domains are a second light chain variable region and the two masking domains are a second heavy chain variable region, wherein the second light chain and heavy chain variable regions form a Two variable domains. The antibody of claim 3 or 10, wherein the second variable domain binds to tissue factor (TF), red blood cells or albumin. The antibody of claim 1, 7 or 8, wherein the masking domain is an albumin binding peptide or protein. A performance vector comprising a coding region controlled by a promoter as claimed in claims 1 to 12. A cell comprising the expression vector of claim 13. A pharmaceutical composition comprising the antibody of claims 1 to 12 formulated with a pharmaceutically acceptable buffer, carrier or diluent. A method of treating a blood coagulation disorder in an individual comprising administering to the individual an amount of an antibody sufficient to promote coagulation in the individual, the antibody comprising: (a) a first variable domain comprising a first light chain and a first heavy chain a variable region, the first variable domain binding to a tissue factor pathway inhibitor (TFPI); (b) a masking domain linked to an amine-based end of the first light chain and/or the first heavy chain variable region; (c) a protease cleavable linker inserted between the first light chain and/or the first heavy chain variable region and the masking domain. The method of claim 16, wherein the protease cleavable domain is a thrombin, plasmin, factor VIIa or factor Xa cleavage site. The method of claim 16, wherein the masking domain comprises a second variable domain comprising a second light chain and a second heavy chain variable region. The method of claim 16, wherein the individual is a human. The method of claim 16, wherein the individual is a non-human mammal. The method of claim 16, wherein the antibody is a single chain antibody. The method of claim 16, wherein the antibody is bivalent and comprises two masking domains, one linked to the amine terminus of each of the first light chain variable regions. The method of claim 16, wherein the antibody is bivalent and comprises two masking domains, one linked to the amine terminus of each of the first heavy chain variable regions. The method of claim 16, wherein the antibody is bivalent and comprises four masking domains, one linked to each of the first light chain variable region and the amine terminal of each of the first heavy chain variable regions. The method of claim 24, wherein the two masking domains are a second light chain variable region and the two masking domains are a second heavy chain variable region, wherein the second light chain and heavy chain variable regions form a Two variable domains. The method of claim 18 or 25, wherein the second variable domain binds to tissue factor (TF), red blood cells or albumin. The method of any one of claims 16, 22 or 23, wherein the masking domain is an albumin binding protein. The method of claim 16, wherein the individual has a trauma, hemophilia or cancer. The method of claim 16, wherein the individual has type A or type B hemophilia. The method of claim 16, wherein the antibody is administered systemically. The method of claim 16, wherein the antibody is administered topically or regionally to the site of bleeding. The method of claim 16, wherein the antibody is administered subcutaneously, intravenously or intraarterially. The method of claim 16, wherein the antibody is IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE antibodies. The method of claim 16, wherein the antibody is a human or a humanized antibody. The method of claim 16, wherein the antibody binds to Kunitz domain 2 of a human tissue factor pathway inhibitor.